Dehydrogenases dependent upon NAD and NADP coenzymes constitute the largest group of redox enzymes known. These enzymes catalyze reactions of the type EQU Substrate+NAD.sup.+ (NADP.sup.+)=Product+H.sup.+ +NADH (NADPH) (1)
These enzymes have been used in only a few amperometric biosensors, partly due to the fact that the electrochemistry of both the oxidized (NAD.sup.+) and reduced (NADH) forms of the coenzyme is very irreversible. The oxidation of NADH results in electrode fouling, while the reduction of NAD is complicated by radical formation and dimerization.
The high overvoltage of approximately 1V (J. Moiroux and P. J. Elving, Analytical Chemistry, Vol. 50, p. 1056 (1978)) required for direct oxidation of NADH brings about several important disadvantages. Because the electrode is highly oxidizing, substrates, products, enzymes, and other components in the test solution can react in an unacceptable manner. For example, reaction products may polymerize at the electrode, resulting in a deactivated surface.
The high overvoltage can be reduced to a large extent by immobilizing mediating structures onto the electrode surface (B. Persson and L. Gorton, Journal of Electroanalytical Chemistry, Vol. 292, p. 115 (1990)). The mediating reactions are generally of the type shown below: EQU NADH (NADPH)+Med.sup.+ .fwdarw.NAD.sup.+ (NADP.sup.+)+MedH (2) EQU MedH.fwdarw.Med.sup.+ +H.sup.+ +2e (3)
In these expressions, Med and MedH refer to the oxidized and reduced forms of the mediator, respectively. To date, all of the effective mediators are based on low molecular weight materials which are at least partially soluble in water. This limits the useful lifetime of an electrode modified with these mediators, as the mediators are free to diffuse away from the electrode surface into solution.